no more PCR amplification after I got rid of a contamination - (Mar/28/2008 )

Hi,

I work on plant microsat and am having issues with the very basic PCR step. I try to amplify a 250bp fragment. Everything was perfect until last monday when I got negative controls that became positive. I have changed all the chemicals, autoclaved the pipettes (at least the parts that can be autoclaved ). Plus, I decreased the number of cycles from 35 to 30. I think I could finally get rid of it.

But now, the amplifications are very weak (while they were strong when the contaminant was there and probably amplified !). This has probably nothing to do with the contamination but I am not very sure though. Has any of of you ever encountered that situation ?
I tried to quantify the genomic DNA I extracted from leaves using the DNeasy plant kit. 260/280 ratio range from 1.2 to 2 (but reach 5 for very few samples !). 260/230 are conversely very bad (0.1 to 0.9). Do you think I should do an ethanol precipitation ?

Thanks !

have you tried to change back the number of cycles? less cycles, less amplification?

For any Dneasy kit:
after the 2nd wash step, discard the elute and centrifuge the column at high speed for dry the membrane (etoh interfere with PCR reaction). In the DNA elution step add 1/2 volume of buffer incubate 3-5 min, centrifuge, repeat with the other 1/2. If concentration still low add the elute again to the membrane and incubate for 10 min at 50C then centrifuge. If still have problems maybe there is no disruption of cells.

hi,

thanks for the replies !

Yes, less cycles (30 instead of 35) leads to less amplification. So I tried again with 35, and now, the negative control is negative on the gel but when I run it on the sequencer there are peaks (same size as the PCR products expected!).